Recognition of foreign DNA in the cytosol of host cells is critical for the initiation of immune responses to DNA viruses. Recognition of viral DNA activates numerous signaling pathways, including the induction of potent antiviral cytokines such as type I interferons. Although the cytosolic DNA-sensing pathway evolved to detect viruses, our recent preliminary data also suggest that DNA-dependent activation of STING may also be critical in the response to tumors. Here we propose to investigate how recognition of cytosolic DNA leads to activation of anti-tumor and anti-viral responses by Natural Killer (NK) cells. Our overall hypothesis is that cytosolic detection of DNA is a common mechanism underlying innate recognition of viruses and tumors by NK cells. We describe three Aims that will illuminate how the cGAS-STING pathway initiates anti-viral and anti-tumor responses. These Aims represent a close collaboration between the Raulet Lab, which has expertise in NK cells and the NKG2D activating receptor, and the lab of consultant Russell Vance, which has expertise in cytosolic DNA sensing and the STING pathway. The Aims are as follows: (1) Determine how the cGAS-STING pathway activates NK cells and other immune cells in vivo. Our preliminary data show that STING engagement by HSV-1 or synthetic 2'3'-cGAMP activates NK cells and other immune cells in vivo. STING activation leads to production of type I IFN, STAT6-dependent chemokines, and activation of NKG2D ligands. The mechanisms linking these pathways to NK activation, and the cell types involved, will be determined in vivo using various knockout mice. (2) Determine if cGAS-STING signaling is required for innate immune control of tumors. Mice harboring an E-myc transgene, which develop B cell lymphomas, will be crossed to mice deficient in the cGAS-STING signaling pathway. We will determine whether loss of cGAS-STING accelerates tumorigenesis in this model. We hypothesize that cGAS-STING signaling is required for tumor cell and possibly APC expression of ligands that activate NK cell receptors (e.g., NKG2D and DNAM1). (3) Determine the mechanisms by which DNA damage leads to accumulation of cytosolic DNA. Our preliminary data indicate that cells experiencing DNA damage or undergoing transformation exhibit increased and aberrant accumulation of cytosolic DNA derived largely from endogenous retroviruses/elements. In this Aim we will assess whether increased cytosolic DNA is due to increased expression of retrovirus/element RNA, and/or increased cytosolic reverse transcriptase activity, and/or decreased activity of cytosolic DNA exonucleases (e.g., TREX1).